Tick-borne bacterial pathogens of humans cause significant morbidity and mortality throughout the United States and abroad. Lyme disease (LD), caused by Borrelia burgdorferi, is the most prevalent arthropod-borne disease of humans in the United States and many other countries throughout Europe and Asia. Tick-borne relapsing fever (TBRF), caused by Borrelia hermsii, is endemic in scattered foci throughout many regions of higher elevation in the western United States. Our work has focused on three areas: 1) to improve on the serodiagnosis of LD and TBRF fever by using recombinant DNA technology to clone genes of spirochetes that express proteins that induce specific and detectable antibody responses; 2) to examine how spirochetes adapt to their tick and mammalian hosts; 3) perform genetic analysis of spirochete isolates and comparative genomics between relapsing fever and Lyme disease spirochetes. This work requires that we maintain colonies of Ixodes scapularis and Ornithodoros hermsi, the respective tick vectors of LD and TBRF spirochetes, and infect these ticks via a laboratory mouse - tick cycle. Our interests in serodiagnosis and adaptations associated with tick infection and transmission demand that we have a solid understanding of genetic and phenotypic variation within our species of interest. Genome sequencing projects of the relapsing fever spirochetes, Borrelia hermsii and Borrelia turicatae, revealed the presence of three open reading frames (ORFs) on the chromosome that contained a high number of repeats encoding proteic motifs: BH0209, BH0512 and BH0553. Comparative genomics of relapsing fever bacteria to the Lyme disease spirochete indicated that the repeated motifs found in the amino acid sequences encoded by the three ORFs were unique in relapsing fever spirochetes. In silico analysis showed that the predicted amino acid sequences encoded by these three genes have highly hydrophobic N-terminal extremities and may be secreted. PCR analyses from the genomic DNA of 28 isolates of B. hermsii and 8 isolates of B. turicatae demonstrated a high degree of polymorphism correlated to the number of repeats in each gene. Real time RT-PCR analysis of RNA produced by B. hermsii showed that the three ORFs are transcribed in vitro at 34?C and 24?C. To assess the synthesis of the three proteins, antisera were generated against recombinant antigens or peptides. Immunoblot analysis using the antisera revealed that BH0512 and BH0553 encoded proteins (P-512 and P-553) were expressed in vitro and in vivo in experimentally infected mice. Further analyses also demonstrated that the P-512 and P-553 were surface-exposed and highly polymorphic. These proteins may play a role in antigenic variation to escape the host's immune response to infection. This year we also completed our analysis of the transmission-associated protein (Vtp, for variable tick protein) in Borrelia hermsii, When argasid ticks, Ornithodoros hermsii, acquire these spirochetes from the blood of small mammals, the bacteria switch their outer surface from one of many bloodstream, variable major proteins (Vmps) to a unique protein, Vtp. Vtp-positive spirochetes persist in the tick?s salivary glands until they are transmitted to the next mammalian host, in which the spirochetes quickly revert to their previous bloodstream Vmp. Vtp may be critical for successful tick transmission of B. hermsii, but the gene encoding this protein has been described previously in only one isolate. Therefore, we described 31 isolates of B. hermsii that originated from localities throughout much of its known geographic distribution. DNA sequence analysis of the genes encoding 16S rRNA, flagellin (FlaB), DNA gyrase subunit B (GyrB) and glycerophosphodiester phosphodiesterase (GlpQ) demonstrated that the isolates separated into two distinct clones designated Genomic Group I and Genomic Group II. The Vtp gene was found and sequenced in all isolates with seven major Vtp types identified. Little or no sequence variation existed within types but between the types significant variation occurred, similar to the pattern of diversity that has been described for the gene encoding outer surface protein C (OspC) in Lyme disease spirochetes. The pattern of sequence relatedness among the Vtp types for all isolates was incongruent in two branches compared to the two genomic groups, which suggested that horizontal transfer and recombination was responsible for some of the variation observed.